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WO2025008000A1 - 一种高效诱导肌肉干细胞的培养基及方法 - Google Patents

一种高效诱导肌肉干细胞的培养基及方法 Download PDF

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WO2025008000A1
WO2025008000A1 PCT/CN2024/113008 CN2024113008W WO2025008000A1 WO 2025008000 A1 WO2025008000 A1 WO 2025008000A1 CN 2024113008 W CN2024113008 W CN 2024113008W WO 2025008000 A1 WO2025008000 A1 WO 2025008000A1
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muscle stem
medium
stem cell
muscle
stem cells
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郭莹
杨一行
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Exoneuglia Biotechnology Suzhou Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • C12N5/0659—Satellite cells
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the technical field of pluripotent stem cell induction, and in particular to a culture medium and a method for efficiently inducing muscle stem cells to obtain muscle fibers or skeletal muscles.
  • Muscles are the most abundant component of the human body and are essential for maintaining body movement and health. Under normal circumstances, if muscles are damaged, the resting muscle stem cells located between the basement membrane and plasma membrane of muscle fibers will be activated, proliferate and differentiate to produce new muscle fibers, and then repair the damaged muscles. Although adult muscle stem cells have strong regenerative potential, progressive muscle necrosis caused by diseases such as aging muscle atrophy and Duchenne muscular dystrophy often leads to a decrease or complete loss of the regenerative repair ability of muscle stem cells, eventually leading to paralysis or even death of the patient.
  • stem cell technology has brought great hope for the treatment of aging or muscle diseases such as DMD, but it still faces a major challenge: the difficulty in isolating, culturing in vitro, and expanding muscle stem cells in large quantities, resulting in an insufficient supply of muscle stem cells, which seriously limits the clinical application of stem cell therapy.
  • one of the purposes of the present invention is to provide a method for efficiently inducing muscle stem cells. This method can obtain a considerable amount of muscle stem cells at one time for treatment and simulation of disease models, which effectively solves the problem of difficulty in obtaining and amplifying human muscle stem cells.
  • the present invention adopts the following technical scheme to achieve the above technical purpose:
  • the present invention provides a method for efficiently inducing muscle stem cells, comprising the following steps:
  • the cells are digested with a cell digestion solution when the cell confluence reaches 60-80%, and then placed on a low-adsorption plate and suspended in a first muscle stem cell induction medium for 1 day; wherein the first muscle stem cell induction medium includes a stem cell basal medium and 5-10 ⁇ M ROCK inhibitor Y-27632;
  • the culture medium is replaced with a second muscle stem cell induction medium for 5 to 7 days to obtain muscle stem cell spheres; wherein the second muscle stem cell induction medium includes a stem cell basal medium and 10 to 15 ⁇ M glycogen synthase kinase-3 inhibitor CHIR99021;
  • the muscle stem cell balls are digested with cell digestion solution and then centrifuged and transferred to adherent culture.
  • a third muscle stem cell induction medium is added to a well plate coated with a collagen solution and cultured for 1 day, then replaced with a muscle stem cell proliferation medium for culture, and expanded for 2 to 5 generations; wherein the third muscle stem cell induction medium includes a stem cell basal medium, 10 to 15 ⁇ M glycogen synthase kinase-3 inhibitor CHIR99021 and 5 to 10 ⁇ M ROCK inhibitor Y-27632;
  • the muscle stem cell proliferation medium includes a cell growth basal medium, bovine serum and basic fibroblast growth factor BFGF, wherein the volume concentration of the bovine serum is 10 to 20%, and the concentration of the basic fibroblast growth factor BFGF is 2 to 5 ng/mL;
  • the original muscle stem cell proliferation medium is discarded, and a muscle stem cell differentiation medium is added to induce differentiation for 6 to 8 days to obtain mature muscle fibers;
  • the muscle stem cell differentiation medium includes mammalian cell growth basal medium, horse serum, tyrosine kinase receptor A agonist and/or prostaglandin synthetase inhibitor, the tyrosine kinase receptor A agonist is amitriptyline with a concentration of 1 to 5 ⁇ M, the prostaglandin synthetase inhibitor is benzydamine with a concentration of 1 to 5 ⁇ M, and the volume proportion of horse serum is 2 to 5%.
  • the method provided by the present invention obtains muscle stem cell balls with higher purity by suspension culture when obtaining mature muscle fibers.
  • the muscle stem cell balls are digested into single cells and then rapidly expanded on a 2D level.
  • a differentiation medium containing specific small molecule inhibitors is used for culture.
  • the pluripotent stem cells can be rapidly induced to obtain muscle stem cells and mature muscle fibers or skeletal muscles, and a considerable amount of muscle stem cells can be obtained at one time for treatment and simulation of disease models, which can well solve the problem of difficulty in obtaining and expanding human muscle stem cells.
  • the basal culture medium in the muscle stem cell differentiation medium is DMEM medium; the basal culture medium in the first muscle stem cell induction medium is mTeSR1 medium, and the basal culture medium in the second and third muscle stem cell induction medium is E6 medium; the basal culture medium in the muscle stem cell proliferation medium is F10 medium.
  • the collagen solution is prepared by adding acetic acid solution to Collagen I Rat Tail liquid.
  • the method for expanding human pluripotent stem cells until they can be induced to differentiate is: growing and maintaining the human pluripotent stem cells on a Matrigel-coated plate in mTeSR1 culture medium.
  • human pluripotent stem cells are obtained by reprogramming human blood monocytes using Episome reprogramming plasmids.
  • the second purpose of the present invention is to protect a culture medium for efficiently inducing muscle stem cells, including the above-mentioned muscle stem cell differentiation culture medium.
  • it also includes the above-mentioned first muscle stem cell induction medium, second muscle stem cell induction medium, third muscle stem cell induction medium and muscle stem cell proliferation medium.
  • the third object of the present invention is to protect a kit for efficiently inducing muscle stem cells, including the above-mentioned culture medium.
  • the method for efficiently inducing muscle stem cells provided by the present invention can obtain a considerable amount of muscle stem cells at one time for treatment and simulation of disease models, solving the problem of difficulty in obtaining and amplifying human muscle stem cells.
  • FIG1 shows the expression level of MF20 marker in muscle fibers obtained after 7 days of induction of differentiation in the examples and comparative examples
  • FIG2 shows the expression of Myogenin markers in muscle fibers obtained after 7 days of induction of differentiation in the Examples and Comparative Examples
  • FIG3 shows the expression level of Dystrophin marker in muscle fibers obtained after 7 days of induction of differentiation in the Examples and Comparative Examples;
  • FIG4 shows the expression levels of PAX7 and PAX3 markers in muscle stem cells obtained after induction of differentiation in Example 1 and Comparative Example 7;
  • Figure 5 shows the observation results of cells under a microscope at different stages in Example 1 of the present invention, wherein a is the observation result of human peripheral blood mononuclear cells under a microscope before reprogramming, b is the observation result of induced pluripotent stem cells cultured under a microscope after 10 generations of reprogramming, c is the observation result of induced pluripotent stem cells when they were transferred to suspension culture, d is the observation result of induced muscle stem cell suspension spheres, e is the observation result of induced muscle stem cell suspension spheres after digestion and adherence culture for 1 day, and f is the observation result of muscle fibers obtained after differentiation under a microscope.
  • the reagents used in the embodiments of the present invention are all commercially available products.
  • the experimental methods in the following embodiments where specific conditions are not specified are usually carried out under conventional conditions, such as those described in "Molecular Cloning: A Laboratory Manual” compiled by Sambrook et al. (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer.
  • This embodiment provides a method for obtaining muscle stem cells, comprising the following steps:
  • iPS Induced pluripotent stem cells
  • iPS Induced pluripotent stem cells
  • the induced pluripotent stem cells were grown and maintained on six-well plates coated with Matrigel (purchased from Corning) in mTeSR1 medium.
  • Induced differentiation amplify induced pluripotent stem cells to the 20th generation and then induce differentiation.
  • degree of fusion reaches about 70%, use cell digestion solution (Accutase, purchased from Stemcell Technologies) to digest for 5 minutes, then put it back into low-adsorption plate for suspension culture, put it on the shaker in the incubator, add the first muscle stem cell induction medium (prepared by adding 5 ⁇ M ROCK inhibitor Y-27632 to mTeSR1 medium) and culture for 24 hours; on the third day, change to the second muscle stem cell induction medium (prepared by adding 10 ⁇ M glycogen synthase kinase-3 inhibitor CHIR99021 to E6 medium) and culture for 6 days to obtain muscle stem cell spheres;
  • the muscle stem cell spheres were digested with cell digestion solution (Accutase) for 5 minutes, and then centrifuged and transferred to adherent culture.
  • cell digestion solution (Accutase) for 5 minutes, and then centrifuged and transferred to adherent culture.
  • Six-well plates coated with collagen solution were added with the third muscle stem cell induction medium (E6 medium supplemented with 10 ⁇ M glycogen synthase kinase-3 inhibitor CHIR99021 and 5 ⁇ M ROCK inhibitor Y-27632) and cultured for 1 day;
  • the preparation of the collagen solution is as follows: take 6.6 mL of Collagen I Rat Tail solution (purchased from Thermo Fisher Scientific), add 0.02 mM acetic acid solution, and dilute to 50 mL to obtain the collagen solution.
  • the muscle stem cell proliferation medium contains 20% fetal bovine serum and 2.5 ng/mL basic fibroblast growth factor (BFGF) in a volume ratio;
  • BFGF basic fibroblast growth factor
  • the muscle stem cell differentiation medium is a high-glucose DMEM medium containing 2% horse serum by volume, 3 ⁇ M tyrosine kinase receptor A agonist amitriptyline, 2.5 ⁇ M prostaglandin synthase inhibitor benzyldamine, and 4% serum substitute (Ultroser Serum Substitute, abbreviated as UG) by volume.
  • This embodiment provides a method for obtaining muscle stem cells, comprising the following steps:
  • iPS Induced pluripotent stem cells
  • iPS Induced pluripotent stem cells
  • the induced pluripotent stem cells were grown and maintained on six-well plates coated with Matrigel (purchased from Corning) in mTeSR1 medium.
  • Induced differentiation amplify induced pluripotent stem cells to the 20th generation and then induce differentiation.
  • degree of fusion reaches about 70%, use cell digestion solution (Accutase, purchased from Stemcell Technologies) to digest for 5 minutes, then put it back into low-adsorption plate for suspension culture, put it on the shaker in the incubator, add the first muscle stem cell induction medium (prepared by adding 5 ⁇ M ROCK inhibitor Y-27632 to mTeSR1 medium) and culture for 24 hours; on the third day, change to the second muscle stem cell induction medium (prepared by adding 10 ⁇ M glycogen synthase kinase-3 inhibitor CHIR99021 to E6 medium) and culture for 6 days to obtain muscle stem cell spheres;
  • the muscle stem cell spheres were digested with cell digestion solution (Accutase) for 5 minutes, and then centrifuged and transferred to adherent culture.
  • cell digestion solution (Accutase) for 5 minutes, and then centrifuged and transferred to adherent culture.
  • Six-well plates coated with collagen solution were added with the third muscle stem cell induction medium (E6 medium supplemented with 10 ⁇ M glycogen synthase kinase-3 inhibitor CHIR99021 and 5 ⁇ M ROCK inhibitor Y-27632) and cultured for 1 day;
  • the preparation of the collagen solution is as follows: take 6.6 mL of Collagen I Rat Tail solution (purchased from Thermo Fisher Scientific), add 0.02 mM acetic acid solution, and dilute to 50 mL to obtain the collagen solution.
  • the muscle stem cell proliferation medium contained 20% fetal bovine serum and 2.5 ng/mL basic fibroblast growth factor (BFGF) by volume.
  • BFGF basic fibroblast growth factor
  • the muscle stem cell differentiation medium contains: 2% horse serum by volume, 3 ⁇ M tyrosine kinase receptor A agonist amitriptyline (Amitriptyline), and 4% serum substitute (Ultroser Serum Substitute, High glucose DMEM medium (abbreviated as UG).
  • This embodiment provides a method for obtaining muscle stem cells, comprising the following steps:
  • iPS Induced pluripotent stem cells
  • iPS Induced pluripotent stem cells
  • the induced pluripotent stem cells were grown and maintained on six-well plates coated with Matrigel (purchased from Corning) in mTeSR1 medium.
  • Induced differentiation amplify induced pluripotent stem cells to the 20th generation and then induce differentiation.
  • degree of fusion reaches about 70%, use cell digestion solution (Accutase, purchased from Stemcell Technologies) to digest for 5 minutes, then put it back into low-adsorption plate for suspension culture, put it on the shaker in the incubator, add the first muscle stem cell induction medium (prepared by adding 5 ⁇ M ROCK inhibitor Y-27632 to mTeSR1 medium) and culture for 24 hours; on the third day, change to the second muscle stem cell induction medium (prepared by adding 10 ⁇ M glycogen synthase kinase-3 inhibitor CHIR99021 to E6 medium) and culture for 6 days to obtain muscle stem cell spheres;
  • the muscle stem cell spheres were digested with cell digestion solution (Accutase) for 5 minutes, and then centrifuged and transferred to adherent culture.
  • cell digestion solution (Accutase) for 5 minutes, and then centrifuged and transferred to adherent culture.
  • Six-well plates coated with collagen solution were added with the third muscle stem cell induction medium (E6 medium supplemented with 10 ⁇ M glycogen synthase kinase-3 inhibitor CHIR99021 and 5 ⁇ M ROCK inhibitor Y-27632) and cultured for 1 day;
  • the preparation of the collagen solution is as follows: take 6.6 mL of Collagen I Rat Tail solution (purchased from Thermo Fisher Scientific), add 0.02 mM acetic acid solution, and dilute to 50 mL to obtain the collagen solution.
  • the muscle stem cell proliferation medium contained 20% fetal bovine serum and 2.5 ng/mL basic fibroblast growth factor (BFGF) by volume.
  • BFGF basic fibroblast growth factor
  • the muscle stem cell differentiation medium is a high-glucose DMEM medium containing 2% horse serum by volume, 2.5 ⁇ M prostaglandin synthase inhibitor benzyldamine (Benzydamine), and 4% serum substitute (Ultroser Serum Substitute, abbreviated as UG) by volume.
  • Comparative Examples 1 to 6 all provide a method for obtaining muscle stem cells, which differ from Examples 1 to 3 in that the muscle stem cell differentiation culture medium is different, wherein:
  • the muscle stem cell differentiation medium in Comparative Example 1 is a high-glucose DMEM medium containing 2% horse serum by volume and 4% serum substitute (Ultroser Serum Substitute, UG for short) by volume;
  • the muscle stem cell differentiation medium in Comparative Example 2 is a high-glucose DMEM medium containing 2% horse serum by volume, 3 ⁇ M ⁇ 1 adrenergic receptor blocker Atenolol, and 4% serum substitute (Ultroser Serum Substitute, referred to as UG) by volume;
  • the muscle stem cell differentiation medium in Comparative Example 3 is a high-glucose DMEM medium containing 2% horse serum, 5 ⁇ M escin, and 4% serum substitute (Ultroser Serum Substitute, UG) by volume;
  • the muscle stem cell differentiation medium in Comparative Example 4 is a high-glucose DMEM medium containing 2% horse serum, 3 ⁇ M Fipexide, and 4% serum substitute (Ultroser Serum Substitute, UG for short) by volume;
  • the muscle stem cell differentiation medium in Comparative Example 5 is a high-glucose DMEM medium containing 2% horse serum, 1 ⁇ M urethane, and 4% serum substitute (Ultroser Serum Substitute, UG) by volume;
  • the muscle stem cell differentiation medium in Comparative Example 6 is a high-glucose DMEM medium containing 2% horse serum, 3 ⁇ M Miltefosine, and 4% serum substitute (Ultroser Serum Substitute, UG for short) in a volume ratio.
  • This comparative example provides a method for obtaining muscle stem cells, which is a conventional method for obtaining muscle stem cells commonly used in the art, and is specifically as follows:
  • iPS Induced pluripotent stem cells
  • iPS Induced pluripotent stem cells
  • the induced pluripotent stem cells were grown and maintained on six-well plates coated with Matrigel (purchased from Corning) in mTeSR1 medium.
  • each marker i.e., the proportion of positive cells
  • the expression of each marker i.e., the proportion of positive cells
  • the expression of each marker i.e., the proportion of positive cells
  • the expression levels of each marker in the treatment groups to which the small molecule compounds Amitriptyline and Benzydamine were added separately and the treatment groups to which the small molecule compounds Amitriptyline and Benzydamine were added together were significantly increased, indicating that the small molecule compounds Amitriptyline and Benzydamine, whether used alone or together, have a promoting effect on the differentiation of muscle stem cells, and in the treatment group to which Amitriptyline and Benzydamine were added simultaneously, the induction efficiency was increased by 1.2 times compared with the treatment group to which Amitriptyline was added alone, and the efficiency of the treatment group to which Benzydamine was added alone was increased by 1.4 times.
  • Figure 5 shows the observation results of cells under a microscope at different stages in Example 1 of the present invention, wherein a is the observation result of human peripheral blood mononuclear cells under a microscope before reprogramming, b is the observation result of induced pluripotent stem cells cultured under a microscope after 10 generations of reprogramming, c is the observation result of induced pluripotent stem cells when transferred to suspension culture, d is the observation result of induced muscle stem cell suspension spheres, e is the observation result of induced muscle stem cell suspension spheres after digestion and adherence culture for 1 day, and f is the observation result of muscle fibers obtained after differentiation under a microscope, indicating that a large number of mature muscle fibers can be successfully obtained using the method described in the present invention.
  • the inventors obtained through further experiments that when the content of ROCK inhibitor Y-27632 in the first muscle stem cell induction medium is 5-10 ⁇ M, the content of glycogen synthase kinase-3 inhibitor CHIR99021 in the second muscle stem cell induction medium is 10-15 ⁇ M, the content of glycogen synthase kinase-3 inhibitor CHIR99021 in the third muscle stem cell induction medium is 10-15 ⁇ M, the content of ROCK inhibitor Y-27632 is 5-10 ⁇ M, the volume concentration of bovine serum in the muscle stem cell proliferation medium is 10-20%, the concentration of basic fibroblast growth factor BFGF is 2-5 ng/mL, the concentration of tyrosine kinase receptor A agonist amitriptyline in the muscle stem cell differentiation medium is 1-5 ⁇ M, the concentration of prostaglandin synthase inhibitor benzydamine is 1-5 ⁇ M, and the volume proportion of horse serum is 2-5%, a considerable amount of muscle fibers can be obtained.

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Abstract

一种高效诱导肌肉干细胞获得肌纤维或骨骼肌的方法。该方法首先是采用悬浮培养及三种肌肉干细胞诱导培养基进行分步诱导，之后采用含有牛血清和碱性成纤维细胞生长因子BFGF的培养基进行扩增，再之后采用含有特定化学小分子的分化培养基进行诱导分化，该方法可以快速的将多能干细胞诱导获得肌肉干细胞及成熟肌纤维或骨骼肌肉，并且一次性即可得到相当量的肌肉干细胞进行治疗及疾病模型的模拟，很好的解决了人源肌肉干细胞获取及扩增难的问题。

Description

一种高效诱导肌肉干细胞的培养基及方法 技术领域
本发明涉及多能干细胞诱导技术领域，具体涉及一种高效诱导肌肉干细胞获得肌纤维或骨骼肌的培养基及方法。
背景技术
肌肉是人体最丰富的组成部分，对维持机体运动和生命健康至关重要。在正常情况下，如果肌肉发生损伤，位于肌肉纤维基底膜和质膜之间的静息状态的肌肉干细胞会被激活，并增殖分化产生新的肌纤维，进而修复受损肌肉。尽管成人肌肉干细胞具有强大的再生潜能，然而，由于老龄化肌肉萎缩和杜氏肌营养不良症等疾病引起的渐进性肌肉坏死，往往会导致肌肉干细胞的再生修复能力下降或者完全丧失，最终导致病人瘫痪，甚至死亡。
近年来，干细胞技术为衰老或DMD等肌肉疾病的治疗带来了巨大希望，但仍旧面临一个重大的挑战是难以实现肌肉干细胞的分离、体外培养和大量扩增，从而导致肌肉干细胞的供应不足，严重限制了干细胞疗法的临床应用。
发明内容
针对现有技术所存在的技术问题，本发明的目的之一在于提供一种高效诱导肌肉干细胞的方法，采用该方法可以一次性得到相当量的肌肉干细胞进行治疗及疾病模型的模拟，很好的解决了人源肌肉干细胞获取及扩增难的问题。
本发明采用如下技术方案来实现上述技术目的：本发明提供一种高效诱导肌肉干细胞的方法，包括以下步骤：
扩增人源多能干细胞至可以诱导分化后，待细胞融合度达到60～80％使用细胞消化液消化，并放入低吸附性板加入第一肌肉干细胞诱导培养基悬浮培养1天；其中，第一肌肉干细胞诱导培养基包括干细胞基础培养基和5～10μM ROCK抑制剂Y-27632；
第3天时，更换培养基为第二肌肉干细胞诱导培养基培养5～7天，获得肌肉干细胞球；其中，第二肌肉干细胞诱导培养基包括干细胞基础培养基和10～15μM糖原合成酶激酶-3抑制剂CHIR99021；
用细胞消化液消化肌肉干细胞球后离心转贴壁培养，用Collagen溶液包被的孔板，加入第三肌肉干细胞诱导培养基培养1天后，换成肌肉干细胞增殖培养基培养，并扩增2～5代；其中，第三肌肉干细胞诱导培养基包括干细胞基础培养基、10～15μM糖原合成酶激酶-3抑制剂CHIR99021和5～10μM ROCK抑制剂Y-27632；肌肉干细胞增殖培养基包括细胞生长基础培养基、牛血清和碱性成纤维细胞生长因子BFGF，其中，牛血清的体积浓度为10～20％，碱性成纤维细胞生长因子BFGF的浓度为2～5ng/mL；
将扩增完成后的肌肉干细胞吸弃原有肌肉干细胞增殖培养基，添加肌肉干细胞分化培养基，诱导分化6～8天，即得成熟肌纤维；其中，肌肉干细胞分化培养基包括哺乳动物细胞生长基础培养基、马血清、酪氨酸激酶受体A激动剂和/或前列腺素合成酶抑制剂，酪氨酸激酶受体A激动剂为阿米替林，浓度为1～5μM，前列腺素合成酶抑制剂为苄达明，浓度为1～5μM，马血清的体积占比为2～5％。
本发明所提供的方法在获得成熟肌纤维时，通过悬浮培养的方式获得纯度更高的肌肉干细胞球，将此肌肉干细胞球消化为单细胞后在2D层面上快速扩增，肌肉干细胞至成熟肌纤维分化中，采用含有特定小分子抑制剂的分化培养基进行培养，可以快速的将多能干细胞诱导获得肌肉干细胞及成熟肌纤维或骨骼肌肉，并且一次性即可得到相当量的肌肉干细胞进行治疗及疾病模型的模拟，能够很好的解决人源肌肉干细胞获取及扩增难的问题。
作为一种优选的实施方式，所述肌肉干细胞分化培养基中的基础培养基 为DMEM培养基；第一肌肉干细胞诱导培养基中的基础培养基为mTeSR1培养基，第二、三肌肉干细胞诱导培养基中的基础培养基为E6培养基；肌肉干细胞增殖培养基中的基础培养基为F10培养基。
作为一种优选的实施方式，所述Collagen溶液由Collagen I Rat Tail液中加入醋酸溶液配成配制而成。
作为一种优选的实施方式，扩增人源多能干细胞至可以诱导分化的方法为：将人源多能干细胞在在mTeSR1培养基中的Matrigel包被的孔板上生长和维持。
作为一种优选的实施方式，人源多能干细胞由人血液单核细胞使用Episome重编程质粒重编程得到。
本发明的目的之二在于保护一种高效诱导肌肉干细胞的培养基，包括上述的肌肉干细胞分化培养基。
作为一种优选的实施方式，还包括上述的第一肌肉干细胞诱导培养基、第二肌肉干细胞诱导培养基、第三肌肉干细胞诱导培养基以及肌肉干细胞增殖培养基。
本发明的目的之三在于保护一种高效诱导肌肉干细胞的试剂盒，包括上述的培养基。
本发明所提供的高效诱导肌肉干细胞的方法可以一次得到相当量的肌肉干细胞进行治疗及疾病模型的模拟，解决了人源肌肉干细胞获取难以及扩增难的问题。
附图说明
图1为实施例和对比例中诱导分化7天后获得的肌纤维中MF20标志物的表达量；
图2为实施例和对比例中诱导分化7天后获得的肌纤维中Myogenin标志物的表达量；
图3为实施例和对比例中诱导分化7天后获得的肌纤维中Dystrophin标志物的表达量；
图4为实施例1和对比例7中诱导分化后获得的肌肉干细胞中PAX7、PAX3标志物的表达量；
图5为本本发明实施例1中细胞不同时期显微镜下的观察结果，其中a为重编程前的人外周血单核细胞显微镜下的观察结果，b为重编程后经过传代10代后的诱导性多能干细胞贴壁培养下显微镜下的观察结果，c为诱导性多能干细胞转悬浮培养时显微镜下的观察结果，d为诱导后的肌肉干细胞悬浮球的观察结果，e为诱导后的肌肉干细胞悬浮球消化后贴壁培养1天后显微镜下的观察结果，f为分化后获得的肌肉纤维在显微镜下的观察结果。
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明，以使本领域的技术人员更加清楚地理解本发明。以下各实施例，仅用于说明本发明，但不用来限制本发明的范围。基于本发明中的具体实施例，本领域普通技术人员在没有做出创造性劳动的情况下，所获得的其他所有实施例，都属于本发明的保护范围。
非特殊说明，本发明实施例采用的试剂均为市售商品。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人编著的《分子克隆：实验室手册》(New York:ColdSpring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
实施例1
本实施例提供了一种获得肌肉干细胞的方法，包括以下步骤：
(1)多能干细胞的获得：诱导性多能干细胞(iPS)由一名健康供体的人血液单核细胞使用Episome重编程质粒(购自赛默飞)在Celetrix EX+电转仪器上重编程获得，其中诱导性多能干细胞在mTeSR1培养基中的基质膜基质(Matrigel，购自康宁)包被的六孔板上生长和维持。
(2)诱导分化：扩增诱导性多能干细胞至20代开始诱导分化，待融合度到70％左右使用细胞消化液(Accutase，购自Stemcell Technologies)消化5分钟，然后重新放入低吸附性板悬浮培养，放到培养箱上的摇床上，加入第一肌肉干细胞诱导培养基(mTeSR1培养基中添加5μM ROCK抑制剂Y-27632制备得到)培养24h；等第三天时，换成第二肌肉干细胞诱导培养基(E6培养基中添加10μM糖原合成酶激酶-3抑制剂CHIR99021制备得到)培养6天，获得肌肉干细胞球；
接下来用细胞消化液(Accutase)消化肌肉干细胞球5分钟，后离心转贴壁培养，用Collagen溶液包被的六孔板，加入第三肌肉干细胞诱导培养基(E6培养基中添加10μM糖原合成酶激酶-3抑制剂CHIR99021和5μMROCK抑制剂Y-27632培养1天；
其中，Collagen溶液的配制如下：取6.6mL Collagen I Rat Tail液(购自赛默飞)加入浓度为0.02mM醋酸溶液，定容至50mL得Collagen溶液。
(3)肌肉干细胞的增殖：第二天更换成肌肉干细胞增殖培养基，扩增2～5代。其中肌肉干细胞增殖培养基为含有体积比为20％的北美胎牛血清和2.5ng/mL碱性成纤维细胞生长因子(简称BFGF)；
(4)将扩增完成后的肌肉干细胞吸弃原有增殖培养基，添加新配制的肌肉干细胞分化培养基，诱导分化七天，即得；其中，肌肉干细胞分化培养基为：含有体积比为2％的马血清、3μM酪氨酸激酶受体A激动剂阿米替林(Amitriptyline)、2.5μM前列腺素合成酶抑制剂苄达明(Benzydamine)、体积比为4％的血清替代物(Ultroser Serum Substitute，简称UG)的高糖DMEM培养基。
实施例2
本实施例提供了一种获得肌肉干细胞的方法，包括以下步骤：
(1)多能干细胞的获得：诱导性多能干细胞(iPS)由一名健康供体的人血液单核细胞使用Episome重编程质粒(购自赛默飞)在Celetrix EX+电转仪器上重编程获得，其中诱导性多能干细胞在mTeSR1培养基中的基质膜基质(Matrigel，购自康宁)包被的六孔板上生长和维持。
(2)诱导分化：扩增诱导性多能干细胞至20代开始诱导分化，待融合度到70％左右使用细胞消化液(Accutase，购自Stemcell Technologies)消化5分钟，然后重新放入低吸附性板悬浮培养，放到培养箱上的摇床上，加入第一肌肉干细胞诱导培养基(mTeSR1培养基中添加5μM ROCK抑制剂Y-27632制备得到)培养24h；等第三天时，换成第二肌肉干细胞诱导培养基(E6培养基中添加10μM糖原合成酶激酶-3抑制剂CHIR99021制备得到)培养6天，获得肌肉干细胞球；
接下来用细胞消化液(Accutase)消化肌肉干细胞球5分钟，后离心转贴壁培养，用Collagen溶液包被的六孔板，加入第三肌肉干细胞诱导培养基(E6培养基中添加10μM糖原合成酶激酶-3抑制剂CHIR99021和5μMROCK抑制剂Y-27632培养1天；
其中，Collagen溶液的配制如下：取6.6mL Collagen I Rat Tail液(购自赛默飞)加入浓度为0.02mM醋酸溶液，定容至50mL得Collagen溶液。
(3)肌肉干细胞的增殖：第二天更换成肌肉干细胞增殖培养基，扩增2～5代。其中肌肉干细胞增殖培养基为含有体积比为20％的北美胎牛血清和2.5ng/mL碱性成纤维细胞生长因子(简称BFGF)。
(4)将扩增完成后的肌肉干细胞吸弃原有增殖培养基，添加新配制的肌肉干细胞分化培养基，诱导分化七天，即得；其中，肌肉干细胞分化培养基为：含有体积比为2％的马血清、3μM酪氨酸激酶受体A激动剂阿米替林(Amitriptyline)、体积比为4％的血清替代物(Ultroser Serum Substitute， 简称UG)的高糖DMEM培养基。
实施例3
本实施例提供了一种获得肌肉干细胞的方法，包括以下步骤：
(1)多能干细胞的获得：诱导性多能干细胞(iPS)由一名健康供体的人血液单核细胞使用Episome重编程质粒(购自赛默飞)在Celetrix EX+电转仪器上重编程获得，其中诱导性多能干细胞在mTeSR1培养基中的基质膜基质(Matrigel，购自康宁)包被的六孔板上生长和维持。
(2)诱导分化：扩增诱导性多能干细胞至20代开始诱导分化，待融合度到70％左右使用细胞消化液(Accutase，购自Stemcell Technologies)消化5分钟，然后重新放入低吸附性板悬浮培养，放到培养箱上的摇床上，加入第一肌肉干细胞诱导培养基(mTeSR1培养基中添加5μM ROCK抑制剂Y-27632制备得到)培养24h；等第三天时，换成第二肌肉干细胞诱导培养基(E6培养基中添加10μM糖原合成酶激酶-3抑制剂CHIR99021制备得到)培养6天，获得肌肉干细胞球；
接下来用细胞消化液(Accutase)消化肌肉干细胞球5分钟，后离心转贴壁培养，用Collagen溶液包被的六孔板，加入第三肌肉干细胞诱导培养基(E6培养基中添加10μM糖原合成酶激酶-3抑制剂CHIR99021和5μMROCK抑制剂Y-27632培养1天；
其中，Collagen溶液的配制如下：取6.6mL Collagen I Rat Tail液(购自赛默飞)加入浓度为0.02mM醋酸溶液，定容至50mL得Collagen溶液。
(3)肌肉干细胞的增殖：第二天更换成肌肉干细胞增殖培养基，扩增2～5代。其中肌肉干细胞增殖培养基为含有体积比为20％的北美胎牛血清和2.5ng/mL碱性成纤维细胞生长因子(简称BFGF)。
(4)将扩增完成后的肌肉干细胞吸弃原有增殖培养基，添加新配制的肌肉干细胞分化培养基，诱导分化七天，即得；其中，肌肉干细胞分化培养基为：含有体积比为2％的马血清、2.5μM前列腺素合成酶抑制剂苄达明(Benzydamine)、体积比为4％的血清替代物(Ultroser Serum Substitute，简称UG)的高糖DMEM培养基。
对比例1～6
对比例1～6均提供一种获得肌肉干细胞的方法，其与实施例1～3的区别均在于肌肉干细胞分化培养基不同，其中，
对比例1中的肌肉干细胞分化培养基为含有体积比为2％的马血清、体积比为4％的血清替代物(Ultroser Serum Substitute，简称UG)的高糖DMEM培养基；
对比例2中的肌肉干细胞分化培养基为含有体积比为2％的马血清、3μMβ1肾上腺素能受体阻断剂阿替洛尔(Atenolol)、体积比为4％的血清替代物(Ultroser Serum Substitute，简称UG)的高糖DMEM培养基；
对比例3中的肌肉干细胞分化培养基为含有体积比为2％的马血清、5μM七叶皂苷(Escin)、体积比为4％的血清替代物(Ultroser Serum Substitute，简称UG)的高糖DMEM培养基；
对比例4中的肌肉干细胞分化培养基为含有体积比为2％的马血清、3μM非哌西特(Fipexide)、体积比为4％的血清替代物(Ultroser Serum Substitute，简称UG)的高糖DMEM培养基；
对比例5中的肌肉干细胞分化培养基为含有体积比为2％的马血清、1μM尿烷(Urethane)、体积比为4％的血清替代物(Ultroser Serum Substitute，简称UG)的高糖DMEM培养基；
对比例6中的肌肉干细胞分化培养基为含有体积比为2％的马血清、3μM米替福新(Miltefosine)、体积比为4％的血清替代物(Ultroser Serum Substitute，简称UG)的高糖DMEM培养基。
对比例7传统诱导方法
本对比例提供一种获得肌肉干细胞的方法，其为本领域常用的传统获得肌肉干细胞的方法，具体如下：
(1)多能干细胞的获得：诱导性多能干细胞(iPS)由一名健康供体的人血液单核细胞使用Episome重编程质粒(购自赛默飞)在Celetrix EX+电转仪器上重编程获得，其中诱导性多能干细胞在mTeSR1培养基中的基质膜基质(Matrigel，购自康宁)包被的六孔板上生长和维持。
(2)诱导分化：当多能干细胞密度达到30％左右时，更换含有10μMCHIR99021、0.5μM LDN193189、10ng/mL bFGF、体积比为1％非必需氨基酸NEAA、体积比为1％ N-2的DMEM培养基诱导三天；更换含有10μMDAPT、20ng/mL bFGF、体积比为1％的非必需氨基酸NEAA、体积比为1％N-2的DMEM培养基诱导两天；更换含有10μM DAPT、10ng/mL HGF、20ng/mL bFGF、体积比为1％非必需氨基酸NEAA、体积比为1％ N-2的DMEM培养基继续诱导两天；更换含有10μM DAPT、10ng/mL HGF、体积比为10％KSR、0.1mMβ-ME、体积比为1％非必需氨基酸NEAA、体积比为1％N-2的DMEM培养基，诱导四天，即获得肌肉干细胞。
结果分析：
(1)在诱导的最后一天，对实施例1～3和对比例1～6中获得的肌纤维进行免疫荧光染色实验，检测MF20、Dystrophin、Myogenin三种成熟肌纤维标志物的表达情况，随后用Image J软件对其荧光程度量化统计，结果如表1及图1～3所示：
表1实施例和对比例中获得的肌纤维中成熟肌纤维标志物表达情况
由上述结果可知，加入分化培养基7天后，添加小分子化合物阿替洛尔(Atenolol)的实验组(即对比例2)中未检测到成熟肌纤维标志物，这说明小分子化合物阿替洛尔(Atenolol)对干细胞具有致死作用；
与不添加小分子化合物的对比例1相比，添加七叶皂苷、非哌西特、尿烷、米替福新的处理组中各标志物表达量(即阳性细胞所占比例)均出现了一定程度的下降，说明小分子化合物七叶皂苷、非哌西特、尿烷、米替福新对干细胞具有轻度抑制分化作用；
与不添加小分子化合物的对比例1相比，分别添加小分子化合物阿米替林(Amitriptyline)和苄达明(Benzydamine)及共同添加小分子化合物阿米替林(Amitriptyline)和苄达明(Benzydamine)的处理组中各标志物表达量均出现了显著提升，说明小分子化合物阿米替林(Amitriptyline)和苄达明(Benzydamine)无论是单独使用或者是共同使用均对肌肉干细胞的分化具有促进作用，并且同时添加阿米替林(Amitriptyline)和苄达明(Benzydamine)的处理组中，其诱导效率比单独添加阿米替林(Amitriptyline)的处理组提高了1.2倍，单独添加苄达明(Benzydamine)的处理组效率提高了1.4倍。
(2)保证起始诱导密度相同，将实施例1中步骤(2)中获得的肌肉干细胞与采用传统方法(对比例7)中获得的肌肉干细胞进行免疫荧光实验，检测PAX7、PAX3两种肌肉干细胞标志物的表达情况，结果如图4和表2所示：
表2实施例1中所述方法和传统方法肌肉干细胞诱导效率比较
由上述结果可知看出，本发明所述的方法肌肉干细胞的诱导效率显著高于传统诱导方法。
图5为本发明实施例1中细胞不同时期显微镜下的观察结果，其中a为重编程前的人外周血单核细胞显微镜下的观察结果，b为重编程后经过传代10代后的诱导性多能干细胞贴壁培养下显微镜下的观察结果，c为诱导性多能干细胞转悬浮培养时显微镜下的观察结果，d为诱导后的肌肉干细胞悬浮球的观察结果，e为诱导后的肌肉干细胞悬浮球消化后贴壁培养1天后显微镜下的观察结果，f为分化后获得的肌肉纤维在显微镜下的观察结果，说明采用本发明所述的方法可成功获得大量成熟的肌纤维。
发明人经过进一步实验得到，当第一肌肉干细胞诱导培养基中ROCK抑制剂Y-27632的含量为5～10μM，第二肌肉干细胞诱导培养基中糖原合成酶激酶-3抑制剂CHIR99021的含量为10～15μM，第三肌肉干细胞诱导培养基中糖原合成酶激酶-3抑制剂CHIR99021的含量为10～15μM，ROCK抑制剂Y-27632的含量为5～10μM，肌肉干细胞增殖培养基中牛血清的体积浓度为10～20％、碱性成纤维细胞生长因子BFGF的浓度为2～5ng/mL，肌肉干细胞分化培养基中酪氨酸激酶受体A激动剂阿米替林浓度为1～5μM，前列腺素合成酶抑制剂苄达明浓度为1～5μM，马血清的体积占比为2～5％，均能获得相当量的肌纤维。
在此有必要指出的是，以上实施例仅限于对本发明的技术方案做进一步的阐述和说明，并不是对本发明的技术方案的进一步的限制，本发明的方法仅为较佳的实施方案，并非用于限定本发明的保护范围。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (8)

  1. 一种高效诱导肌肉干细胞的方法，其特征在于，包括以下步骤：
    扩增人源多能干细胞至可以诱导分化后，待细胞融合度达到60～80％使用细胞消化液消化，并放入低吸附性板加入第一肌肉干细胞诱导培养基悬浮培养1天；其中，第一肌肉干细胞诱导培养基包括干细胞基础培养基和5～10μM ROCK抑制剂Y-27632；
    第3天时，更换培养基为第二肌肉干细胞诱导培养基培养5～7天，获得肌肉干细胞球；其中，第二肌肉干细胞诱导培养基包括干细胞基础培养基和10～15μM糖原合成酶激酶-3抑制剂CHIR99021；
    用细胞消化液消化肌肉干细胞球后离心转贴壁培养，用Collagen溶液包被的孔板，加入第三肌肉干细胞诱导培养基培养1天后，换成肌肉干细胞增殖培养基培养，并扩增2～5代；其中，第三肌肉干细胞诱导培养基包括干细胞基础培养基、10～15μM糖原合成酶激酶-3抑制剂CHIR99021和5～10μM ROCK抑制剂Y-27632；肌肉干细胞增殖培养基包括细胞生长基础培养基、牛血清和碱性成纤维细胞生长因子BFGF，其中，牛血清的体积浓度为10～20％，碱性成纤维细胞生长因子BFGF的浓度为2～5ng/mL；
    将扩增完成后的肌肉干细胞吸弃原有肌肉干细胞增殖培养基，添加肌肉干细胞分化培养基，诱导分化6～8天，即得成熟肌纤维；其中，肌肉干细胞分化培养基包括哺乳动物细胞生长基础培养基、马血清、酪氨酸激酶受体A激动剂和/或前列腺素合成酶抑制剂，酪氨酸激酶受体A激动剂为阿米替林，浓度为1～5μM，前列腺素合成酶抑制剂为苄达明，浓度为1～5μM，马血清的体积占比为2～5％。
  2. 根据权利要求1所述的高效诱导肌肉干细胞的方法，其特征在于，所述肌肉干细胞分化培养基中的基础培养基为DMEM培养基；和/或第一肌肉干细胞诱导培养基中的基础培养基为mTeSR1培养基，第二、三肌肉干细胞诱导培养基中的基础培养基为E6培养基；和/或肌肉干细胞增殖培养基中的基础培养基为F10培养基。
  3. 根据权利要求1所述的高效诱导肌肉干细胞的方法，其特征在于，所述Collagen溶液由Collagen I Rat Tail液中加入醋酸溶液配成配制而成。
  4. 根据权利要求1所述的高效诱导肌肉干细胞的方法，其特征在于，扩增人源多能干细胞至可以诱导分化的方法为：将人源多能干细胞在mTeSR1培养基中的Matrigel包被的孔板上生长和维持。
  5. 根据权利要求1所述的高效诱导肌肉干细胞的方法，其特征在于，人源多能干细胞由人血液单核细胞使用Episome重编程质粒重编程得到。
  6. 一种高效诱导肌肉干细胞的培养基，其特征在于，包括权利要求1中所述的肌肉干细胞分化培养基。
  7. 根据权利要求6所述的高效诱导肌肉干细胞的培养基，其特征在于，还包括权利要求1中所述的第一肌肉干细胞诱导培养基、第二肌肉干细胞诱导培养基、第三肌肉干细胞诱导培养基以及肌肉干细胞增殖培养基。
  8. 一种高效诱导肌肉干细胞的试剂盒，其特征在于，包括权利要求6～7任一项所述的培养基。
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